Ella Nukarinen

Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

The low energy signaling network

Stress impacts negatively on plant growth and crop productivity, caicultural production worldwide. Throughout their life, plants are often confronted with multiple types of stress that affect overall cellular energy status and activate energy-saving responses. The resulting low energy syndrome (LES) includes transcriptional, translational, and metabolic reprogramming and is essential for stress adaptation. The conserved kinases sucrose-non-fermenting-1-related protein kinase-1 (SnRK1) and target of rapamycin (TOR) play central roles in the regulation of LES in response to stress conditions, affecting cellular processes and leading to growth arrest and metabolic reprogramming. We review the current understanding of how TOR and SnRK1 are involved in regulating the response of plants to low energy conditions. The central role in the regulation of cellular processes, the reprogramming of metabolism, and the phenotypic consequences of these two kinases will be discussed in light of current knowledge and potential future developments.

A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms

Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

Protein sumoylation and phosphorylation intersect in Arabidopsis signaling

Summary Conjugation of the small ubiquitin‐related modifier (SUMO) to protein substrates has an impact on stress responses and on development. We analyzed the proteome and phosphoproteome of mutants in this pathway. The mutants chosen had defects in SUMO ligase SIZ1, which catalyzes attachment of single SUMO moieties onto substrates, and in ligases PIAL1 and PIAL2, which are known to form SUMO chains. A total of 2657 proteins and 550 phosphopeptides were identified and quantified. Approximately 40% of the proteins and 20% of the phosphopeptides showed differences in abundance in at least one of the analyzed genotypes, demonstrating the influence of SUMO conjugation on protein abundance and phosphorylation. The data show that PIAL1 and PIAL2 are integral parts of the SUMO conjugation system with an impact on stress response, and confirm the involvement of SIZ1 in plant defense. We find a high abundance of predicted SUMO attachment sites in phosphoproteins (70% versus 40% in the total proteome), suggesting convergence of phosphorylation and sumoylation signals onto a set of common targets.

Integrative molecular profiling indicates a central role of transitory starch breakdown in establishing a stable C/N homeostasis during cold acclimation in two natural accessions of Arabidopsis thaliana

BackgroundThe variation of growth and cold tolerance of two natural Arabidopsis accessions, Cvi (cold sensitive) and Rschew (cold tolerant), was analysed on a proteomic, phosphoproteomic and metabolomic level to derive characteristic information about genotypically distinct strategies of metabolic reprogramming and growth maintenance during cold acclimation.ResultsGrowth regulation before and after a cold acclimation period was monitored by recording fresh weight of leaf rosettes. Significant differences in the shoot fresh weight of Cvi and Rschew were detected both before and after acclimation to low temperature. During cold acclimation, starch levels were found to accumulate to a significantly higher level in Cvi compared to Rschew. Concomitantly, statistical analysis revealed a cold-induced decrease of beta-amylase 3 (BAM3; AT4G17090) in Cvi but not in Rschew. Further, only in Rschew we observed an increase of the protein level of the debranching enzyme isoamylase 3 (ISA3; AT4G09020). Additionally, the cold response of both accessions was observed to severely affect ribosomal complexes, but only Rschew showed a pronounced accumulation of carbon and nitrogen compounds. The abundance of the Cold Regulated (COR) protein COR78 (AT5G52310) as well as its phosphorylation was observed to be positively correlated with the acclimation state of both accessions. In addition, transcription factors being involved in growth and developmental regulation were found to characteristically separate the cold sensitive from the cold tolerant accession. Predicted protein-protein interaction networks (PPIN) of significantly changed proteins during cold acclimation allowed for a differentiation between both accessions. The PPIN revealed the central role of carbon/nitrogen allocation and ribosomal complex formation to establish a new cold-induced metabolic homeostasis as also observed on the level of the metabolome and proteome.ConclusionOur results provide evidence for a comprehensive multi-functional molecular interaction network orchestrating growth regulation and cold acclimation in two natural accessions of Arabidopsis thaliana. The differential abundance of beta-amylase 3 and isoamylase 3 indicates a central role of transitory starch degradation in the coordination of growth regulation and the development of stress tolerance. Finally, our study indicates naturally occurring differential patterns of C/N balance and protein synthesis during cold acclimation.

Boron nitride as desalting material in combination with phosphopeptide enrichment in shotgun proteomics.

Hydrophilic peptides in shotgun proteomics have been shown to be problematic in conventional chromatography. Typically, C18 solid phase extraction or peptide traps are used for desalting the sample prior to mass spectrometry analysis, but the capacity to retain hydrophilic peptides is not very high, causing a bias toward more hydrophobic peptides. This is particularly problematic in phosphoproteomic studies. We tested the compatibility of commercially available boron nitride as a novel material for peptide desalting. Boron nitride can be used to recover a wide range of peptides with different physicochemical properties comparable to combined C18 and graphite carbon material.

The SnRK1 kinase as central mediator of energy signalling between different organelles

SnRK1 is a central integrator of energy signaling in different subcellular locations with emerging roles in organellar and hormone metabolism.

Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals

The evolutionarily highly conserved SNF1‐related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth‐ and stress response‐related metabolic pathways. While the regulation of the mammalian [AMP‐activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well‐characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T‐loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Sumoylation and phosphorylation: hidden and overt links

Post-translational modifications are essential mediators between stimuli from development or the environment and adaptive transcriptional patterns. Recent data allow a first glimpse at how two modifications, phosphorylation and sumoylation, act interdependently to modulate stress responses. In particular, many components of the SUMO conjugation system are phosphoproteins, and some regulators and enzymes of protein phosphorylation can be sumoylated. Equally important, however, a number of proteins can be subject to both modifications. These substrates also have the capacity to connect stimuli transmitted via sumoylation with those transmitted via phosphorylation. As a prime example, we review data suggesting that nitrate reductase is a hub that integrates cues from these two modifications. Powerful proteomics approaches allowed the identification of additional common substrates, paving the way for studies to understand, on a broader basis, the cross-talk of phosphorylation with sumoylation and how it contributes to plant growth.